The primary objective of the proposed research is to develop a detailed understanding of the molecular events of lipoprotein lipase-catalyzed reactions. It is anticipated that such an understanding will facilitate eventual design of activators and inhibitors of lipoproteins lipase (LpL), which can be used for in vivo manipulation of the serum levels of HDL, which may protect against cardiovascular disease, and LDL, which may dispose toward cardiovascular disease. A range of reaction kinetic and biochemical modification probes will be used to illuminate features of the LpL mechanism. Potential transition state analog inhibitors of LpL-catalyzed hydrolysis of water-soluble and lipid-soluble p-nitrophenyl esters will be tested as probes of the structure of high-energy catalytic intermediates. Active site-directed irreversible inhibition will be investigated to provide a basis for identification of amino acids involved in catalysis. pH-Rate and kinetic solvent deuterium isotope effect probes will supply additional information on the identity of active site amino acids, and on the role they play in reaction dynamics and transition state stabilization. LpL-catalyzed hydrolysis of water-soluble p-nitrophenyl esters at subzero temperatures (cryoenzymology) will be studied in an attempt to observe acylenzyme intermediates by UV-visible spectrophotometry. The mechanism by which apolipoprotein C-II stiumlates LpL-catalyzed hydrolysis of lipid-soluble substrates will be studied by determining the effect of apolipoprotein C-II on transition state structure, on LpL fatty acyl specificity and on the sensitivity of LpL to transition state analog inhibition. A general mechanism for apolipoprotein C-II stimulation of LpL-catalyzed hydrolysis of long acyl chain lipid-soluble substrates is introducted in this proposal, and experiments designed to test the validity of the model are suggested.